F2 derivatives as antibacterial agents

ABSTRACT

A compound or its pharmaceutically acceptable salt, as well as a pharmaceutical, composition containing that compound or salt dissolved or dispersed in a pharmaceutically acceptable carrier, and a method of using that compound or salt in an antibacterial treatment. A contemplated compound corresponds in structure to structural Formula I or a pharmaceutically acceptable salt of that compound, wherein V is O or NR 9 , Y is halogen, OR 10 , C 1 -C 4  hydrocarbyl or NHR 10 , Z is NR 2 —X—R 1  or CH 2 —R 8 , n is 1-6, X is H, S(O) 2 , C(O), C(O)NR 7 , C(NH)NR 7  or C(O)O, and R 1 , R 2 , R 7 , R 8 , R 9  and R 10  are defined within.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority of provisional application Ser. No.61/622,163 filed on Apr. 10, 2012, and whose disclosures areincorporated by reference.

FIELD OF INVENTION

The invention relates to antibacterial agents and to their use. Moreparticularly, the invention relates toN-(1-(2-aminoethyl)-1H-tetrazol-5-yl)-3-chlorobenzamide and its amido,sulfonamide, urea and urethane derivatives, their use in the preparationof a pharmaceutical composition and as a bactericide and bacteriostat.

BACKGROUND ART

It is estimated that 20% of newly synthesized proteins are degraded bythe proteosome due to transcriptional or translational errors [Wickneret al., Science 286:1888-1893 (1999)] and this number increases understress conditions such as heat shock [Wickner et al., Science286:1888-1893 (1999); Ingmer et al., Res Microbiol 160:704-710 (2009)].Bacterial proteosomes also control the half-life of transcriptionfactors and rate-limiting enzymes thereby exerting a regulatory effecton gene expression and metabolism. Thus, regulated proteolysis iscritical from a quality control as well as a regulatory standpoint andloss of these intracellular proteases can have detrimental effects[Frees et al., Mol Microbiol 63:1285-1295 (2007); Jenal et al., CurrOpin Microbiol 6:163-172 (2003)]

During infection, pathogens face numerous stress conditions includingnutrient deprivation, exposure to reactive oxygen species, temperatureand pH changes. Loss of the caseinolytic protease (Clp) systemattenuates virulence in several pathogens including B. anthracis and S.aureus [Frees et al., Mol Microbiol 63:1285-1295 (2007); Ingmer et al.,Res Microbiol 160:704-710 (2009); McGillivray et al., J Innate Immun1:494-506 (2009)] making the ClpXp protease a potential target forpharmacological intervention.

Caseinolytic proteases (Clp; EC 3.4.21.92) are endopeptidase enzymes ofpeptidase family S14 originally obtained from bacteria. Clp enzymescontain subunits of two types, ClpP, with peptidase activity, and ClpAor ClpX, that exhibit ATPase activity, autonomous chaperone activity andcan catalyze protein unfolding

These enzymes are intracellular proteases that regulate protein qualityand turnover through controlled proteolysis. Degraded proteins includedamaged or non-functional proteins as well as transcriptionalregulators, rate-limiting enzymes, and proteins tagged duringtrans-translation [Frees et al., Microbiol 63:1285-1295 (2007); Ingmeret al., Res Microbiol 160:704-710 (2009); Keiler et al., Annu RevMicrobiol 62:133-151 (2008)]. The enzymes hydrolyze proteins to smallpeptides in the presence of ATP and Mg²⁺. α-Casein is the usual testsubstrate. In the absence of ATP, only oligopeptides shorter than fiveresidues are hydrolyzed.

Clp protease proteolytic core, ClpP is paired with a regulatory ATPasesuch as CLpA or ClpX. Clp ATPases recognize, unfold and transfer theproteins to ClpP for degradation. Orthologs of ClpXp are found in manybacterial species and are often associated with cellular stress such asheat shock, nutrient deprivation, and oxidative stress [Frees et al.,Microbiol 63:1285-1295 (2007); Ingmer et al., Res Microbiol 160:704-710(2009)]. ClpX and/or ClpP have also been implicated in virulence ofseveral pathogens including Listeria monocytogenes, Salmonella,Staphylococcus aureus, and Streptococcus pneumoniae [Frees et al.,Microbiol 63:1285-1295 (2007); Ingmer et al., Res Microbiol 160:704-710(2009)].

The trans-translation mechanism is a key component of multiple qualitycontrol pathways in bacteria that ensure proteins are synthesized withhigh fidelity in spite of challenges such as transcription errors, mRNAdamage, and translational frameshifting. trans-Translation is performedby a ribonucleoprotein complex composed of tmRNA, a specialized RNA withproperties of both a tRNA and an mRNA, and the small protein SmpB.tmRNA-SmpB interacts with translational complexes stalled at the 3′ endof an mRNA to release the stalled ribosomes and target the nascentpolypeptides and mRNAs for degradation. In addition to quality controlpathways, some genetic regulatory circuits use trans-translation tocontrol gene expression. Diverse bacteria require trans-translation whenthey execute large changes in their genetic programs, includingresponding to stress, pathogenesis, and differentiation.

The compound F2, below, was identified as

part of a high-throughput screen for inhibitors of the protein-taggingand trans-translation degradation pathway in E. coli. F2 has been foundto inhibit the activity of ClpXp protease of bacterial cells withminimal host cell cytotoxicity [McGillivray et al., J Innate Immun1:494-506 (2009)]. Although it is unclear exactly how F2 inhibits theClpXp protease, the data indicate that inclusion of F2 decreases theproteolysis of ClpXp substrates in vivo. Similar to a genetic loss ofClpX, co-treatment of B. anthracis with F2 increased susceptibility ofthe bacteria to cathelicidin antimicrobial peptides. A similar effectwas seen with both methicillin-susceptible and methicillin-resistantstrains of S. aureus suggesting ClpXp also plays a role in cathelicidinresistance in S. aureus. The McGillivray et al. paper is the first studyto show that inhibition of ClpXp could result in a synergisticinteraction with innate immune defenses.

In that paper, McGillivray and co-workers demonstrated that ClpX iscritical for the pathogenesis of Bacillus anthracis, a Gram-positivebacterium that is the causative agent of anthrax [J Innate Immun1:494-506 (2009)]. Loss of ClpX increased susceptibility to innate hostdefenses including cationic antimicrobial peptides and severelyattenuated B. anthracis virulence even in the fully pathogenic Amesstrain. Ibid.

Although McGillivray et al. focused upon cathelicidins in that study,they had previously shown loss of ClpX renders B. anthracis moresensitive to other antimicrobial proteins, including defensins andlysozyme [McGillivray et al., J Innate Immun 1:494-506 (2009)].Therefore, pharmacological inhibition of ClpXp may increasesusceptibility to multiple host defenses.

F2 also sensitizes B. anthracis and S. aureus to antibiotics such aspenicillin and daptomycin, although the synergistic effect between F2and antibiotics was more pronounced in B. anthracis. That enhancementmay reflect the assay conditions or it may indicate that the extent towhich ClpXp influences susceptibility to cell-envelope antibioticsdiffers among species.

Evidence of a connection between Clp proteases and cell-wall actingantibiotics has been seen in other bacterial species. Loss of ClpP inStreptococcus mutans rendered the bacteria more susceptible to the cellwall acting antibiotics bacitracin, polymyxin B, and vancomycin,although no effect was seen with non-cell wall acting antibiotics[Chattoraj et al., J Bacteriol 192:1312-1323 (2010)].

In Mycobacterium tuberculosis, loss of the ClpCP protease, anotherATPase chaperone, resulted in increased susceptibility to cell wallstress induced by vancomycin or SDS [Barik et al., Mol Microbiol75:592-606 (2010)]. Daptomycin is believed to function by membranedepolarization but a recent study demonstrated that it also induces thecell wall stress regulon including Clp family members in S. P aureus[Muthaiyan et al., Antimicrob Agents Chemother 52:980-990 (2008)].

The importance of the ClpXp system is further highlighted by thoseworkers' observation that inhibition increased susceptibility todaptomycin in otherwise non-susceptible strains. However, thissuppression of resistance was only partial, indicating thatnon-Clp-dependent effects probably also contribute to daptomycinresistance. It is likely that loss of ClpXp has pleiotropic effects onthe bacterial cell because the Clp protease regulates a wide range ofgenes [Michel et al., J Bacteriol 188:5783-5796 (2006); Robertson etal., J Bacteriol 184:3508-3520 (2002)].

Cell wall active agents are believed to increase damaged or mis-foldedproteins and result in induction of genes involved in protein turnoversuch as chaperones and proteases [[Muthaiyan et al., Antimicrob AgentsChemother 52:980-990 (2008); Utaida et al., Microbiology 149:2719-2732(2003)]. Loss of ClpXp could hamper this response. The ClpXp proteasemay also be regulating critical components of the cell wall. In E. coli,ClpXp can degrade FtsZ, a major cytoskeletal protein that is implicatedin cell division and cell wall synthesis [Camberg et al., Proc Natl AcadSci USA 106:10614-10619 (2009)]. In B. subtilis, MurAA, an enzymeimportant in peptidoglycan formation, is degraded by ClpCP [Kock et al.,Mol Microbiol 51:1087-1102 (2004)].

It is also possible that cell charge is affected by loss of ClpXp.Daptomycin is an anionic compound that associates with calcium to form acationic complex similar to an antimicrobial peptide. Resistance todaptomycin and cationic antimicrobial peptides has been linked tomutations in mprF (lysine addition to cell membrane phosphatidylglycerol) and the dltABCD operon (alanylation of cell wall teichoicacids) that result in increased net positive surface charge in both S.aureus and B. anthracis [Fisher et al., J Bacteriol 188:1301-1309(2006); Kraus et al., Curr Top Microbiol Immunol 306:231-250 (2006);Samant et al., J Bacteriol 191:1311-1319 (2009); Yang et al., AntimicrobAgents Chemother 53:2636-2637 (2009)].

Consistent with this hypothesis, increasing daptomycin resistance in S.aureus was accompanied by increased resistance to cationic antimicrobialpeptides such as alpha-defensin HNP-1 and platelet microbicidal proteins[Jones et al., Antimicrob Agents Chemother 52:269-278 (2008)]. The Clpprotease could be regulating expression either directly or indirectly ofthe mprF or the dltABCD operon although this has not yet beendemonstrated.

The ClpXp protease is a promising target for pharmacologicalintervention. Inclusion of F2 increased the effectiveness of bacterialkilling by human whole blood indicating this compound can augment innateimmune defenses. This therapeutic effect may be magnified at tissuesites of infection where high levels of antimicrobial peptides areproduced by cells such as keratinocytes, and in patients receivingconcurrent antibiotic therapy with cell wall active agents for whichClpXp inhibition also provides synergism.

Although McGillivray et al. focused on inhibition of ClpXp, uncontrolledactivation of ClpP through a new class of antibiotics, acyldepsipeptides, also has lethal consequences for several bacterialspecies tested [Brotz-Oesterhelt et al., Nat Med 11:1082-1087 (2005)].However, as was seen with acyl depsipeptides [Brotz-Oesterhelt et al.,Nat Med 11:1082-1087 (2005)] and other antimicrobial compounds,potential for bacterial resistance to F2 exists. The mechanism behindthis resistance is at this point unclear, but it may necessitate that F2or another pharmacological inhibitor of ClpXp be used in a combinationrather than single therapy to limit resistance. Nevertheless, the use ofClp inhibitors can be predicted to contribute to antimicrobial activityon multiple levels, through increased susceptibility to innate immunedefenses and decreased resistance to traditional antibiotics,potentially increasing therapeutic effectiveness.

An alternative strategy in antimicrobial therapy is to target andinactivate bacterial virulence factors rather than directly targetinggrowth or survival in the manner of traditional antibiotics [Cegelski etal., Nat Rev Microbiol 6:17-27 (2008)]. Inhibition of virulence factorsinvolved in disease progression should enhance the ability of the hostimmune system to clear the pathogen. The ClpXp protease is a promisingtarget for pharmacological inhibition due to its conserved nature andits role in the virulence of a wide-variety of pathogens.

A present inventor and colleagues have identified several inhibitors ofClpXp using a screening system devised in E. coli [Cheng et al., ProteinSci 16:1535-1542 (2007)]. The ClpXp protease of the Gram positive B.anthracis Sterne and the Gram positive human pathogen, Staphylococcusaureus were targeted using the F2 inhibitor. It was found that F2renders both B. anthracis Sterne and drug-resistant strains of S. aureusmore susceptible to host antimicrobial peptides as well as antibioticsthat target the bacterial cell envelope including the cell wall and/orcell membrane.

BRIEF SUMMARY OF THE INVENTION

The present invention contemplates a compound, as well as apharmaceutical composition containing that compound. Also contemplatedis a method of using that compound as a bacteriostatic or bactericidaltreatment for bacteria, and particularly for Gram positive bacteria suchas B. anthracis, B. subtilis, S. mutans, M. tuberculosis and S. aureus.

A contemplated compound of the invention corresponds in structure tostructural Formula I or a pharmaceutically acceptable salt of thatcompound

wherein V is O or NR⁹, Y is halogen, OR¹⁰, C₁-C₄ hydrocarbyl or NHR¹⁰, Zis NR²—X—R¹ or CH₂—R⁸, and n is a numeral that is 1-6. In a compound ofFormula I, X is hydrido (H), S(O)₂, C(O), C(O)NR⁷, C(NH)NR⁷ or C(O)O,with the proviso that when X is H, R¹ and CH₂—R⁸ are absent. Preferably,X is other than H, and is S(O)₂ or C(O). R⁹ is hydrido (H) or C₁-C₄hydrocarbyl; and R¹⁰ is hydrido or C₁-C₄ hydrocarbyl. R¹ and R⁸ are thesame or different and are an aliphatic, aromatic or heteroaromatic ringsystem containing one ring or two fused rings each having 5-7 atoms inthe ring. The ring system contains up to three substituents other thanhydrogen that themselves can be the same or different (R^(1a), R^(1b),and R^(1c)). Each of those three groups, R^(1a-c), is separatelyselected from the group consisting of C₁-C₆ hydrocarbyl, C₁-C₆hydrocarbyloxy, C₁-C₆ hydrocarbyloxycarbonyl, trifluoromethyl (—OCF₃),trifluoromethoxy (—OCF₃), C₁-C₇ hydrocarboyl (acyl), hydroxy-, halogen,halogen-substituted C₁-C₇ hydrocarboyl, C₁-C₆ hydrocarbylsulfonyl, C₁-C₆hydrocarbyloxysulfonyl, nitro, phenyl, benzyl, cyano, carboxyl, C₁-C₇hydrocarbyl carboxylate [C(O)O—C₁-C₇ hydrocarbyl], carboxamide[C(O)NR³R⁴] or sulfonamide [S(O)₂NR³R⁴] wherein the amido nitrogen ineither group has the formula NR³R⁴ in which R³ and R⁴ are the same ordifferent and are H, C₁-C₄ hydrocarbyl, or R³ and R⁴ together with thedepicted nitrogen form a 5-7-membered ring that optionally contains 1 or2 additional hetero atoms that independently are nitrogen, oxygen orsulfur, MAr, where M is —CH₂—, —O— or —N═N— and Ar is a single-ringedaryl group, and NR⁵R⁶ wherein R⁵ and R⁶ are the same or different andare H, C₁-C₄ hydrocarbyl, C₁-C₄ acyl, C₁-C₄ hydrocarbylsulfonyl, or R⁵and R⁶ together with the depicted nitrogen form a 5-7-membered ring thatoptionally contains 1 or 2 additional hetero atoms that independentlyare nitrogen, oxygen or sulfur. R² and R⁷ are the same or different andare hydrido (H) or C₁-C₄ hydrocarbyl.

A preferred compound of Formula I is a compound that corresponds instructure to structural Formula II, below, or a pharmaceuticallyacceptable salt of that compound

in which V, X, Y, n, R¹ and R² are as defined above. Thus, in FormulaII, X is S(O)₂, C(O), C(O)NR⁷, C(NH)NR⁷ or C(O)O. R¹ is an aliphatic,aromatic or heteroaromatic ring system containing one ring or two fusedrings each having 5-7 atoms in the ring. The ring system contains up tothree substituents other than hydrogen that themselves can be the sameor different (R^(1a), R^(1b), and R^(1c)). Each of those three groups,R^(1a-c), is separately selected from the group consisting of C₁-C₆hydrocarbyl, C₁-C₆ hydrocarbyloxy, C₁-C₆ hydrocarbyloxycarbonyl,trifluoromethyl (—CF₃), trifluoromethoxy (—OCF₃), C₁-C₇ hydrocarboyl(acyl), hydroxy-, halogen, halogen-substituted C₁-C₇ hydrocarboyl, C₁-C₆hydrocarbylsulfonyl, C₁-C₆ hydrocarbyloxysulfonyl, nitro, phenyl, cyano,carboxyl, C₁-C₇ hydrocarbyl carboxylate [C(O)O—C₁-C₇ hydrocarbyl],carboxamide [C(O)NR³R⁴] or sulfonamide [S(O)₂NR³R⁴] wherein the amidonitrogen in either group has the formula NR³R⁴ in which R³ and R⁴ arethe same or different and are H, C₁-C₄ hydrocarbyl, or R³ and R⁴together with the depicted nitrogen form a 5-7-membered ring thatoptionally contains 1 or 2 additional hetero atoms that independentlyare nitrogen, oxygen or sulfur, MAr, where M is —CH₂—, —O— or —N═N— andAr is a single-ringed aryl group, and NR⁵R⁶ wherein R⁵ and R⁶ are thesame or different and are H, C₁-C₄ hydrocarbyl, C₁-C₄ acyl, C₁-C₄hydrocarbylsulfonyl, or R⁵ and R⁶ together with the depicted nitrogenform a 5-7-membered ring that optionally contains 1 or 2 additionalhetero atoms that independently are nitrogen, oxygen or sulfur. R² andR⁷ are the same or different and are hydrido (H) or C₁-C₄ hydrocarbyl.

In preferred practice, n is 2-4. Most preferably n is 2 so that aparticularly preferred compound corresponds in structure to Formula IIA,below, wherein X is other than, R¹ and R² are as defined above

A pharmaceutical composition that contains an antibacterial amount andpreferably an antibacterial amount of a compound or its pharmaceuticallyacceptable salt of Formula I, above, dissolved or dispersed in apharmaceutically acceptable. diluent is also contemplated.

A method of inhibiting the growth of bacteria is another aspect of theinvention. That method contemplates the steps of contacting the bacteriawith an antibacterial amount of a compound of Formula I or itspharmaceutically acceptable salt. In some embodiments, the contactedbacterium is B. anthracis, whereas in other embodiments the bacterium isB. subtilis, S. aureus, M. tuberculosis and S. mutans or yet anotherpreferably Gram positive bacterium. In other embodiments, Gram negativebacteria are the contemplated targets.

In some embodiments, the bacteria are present in a cell culture, whereasin other embodiments, the bacteria are present in an infected mammal andthe bacteria are contacted by administration of the compound to theinfected mammal. Typically, and preferably when the bacteria are presentin an infected mammal, the bacteria are contacted a plurality of times.

Also contemplated is another method of inhibiting the growth ofbacteria, both Gram positive and Gram negative bacteria. In this method,the bacteria are contacted with a synergistic amount of a compoundFormula I or its pharmaceutically acceptable salt and also a synergisticamount of a) a human cathelicidin antimicrobial peptide LL-37 or b) anantibiotic that targets the cell wall and/or the cell membrane.

A particularly preferred compound of Formulas I and II has structuralFormula III, below.

A pharmaceutically acceptable salt of a compound of Formula III is alsocontemplated. A compound of Formula III is often referred to herein asDansyl-F2.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a part of this disclosure,

FIG. 1, in two panels as FIG. 1A and FIG. 1B, illustrate dose responseanalyses of F2 (FIG. 1A) and Dansyl-F2 (FIG. 1B; Formula III) against B.anthracis constructs and S. flexneri. Strains: WT B. anthracis (opencircles); ΔclpX (×); AclpX+pclpX (filled circles); WT+pUTE29 (emptyvector, filled squares); ΔclpX+pUTE29 (open triangles); S. flexneri(open squares).

FIG. 2, in two panels as FIG. 2A and FIG. 2B, illustrate a minimuminhibitory concentration (MIC) determination of F2 (FIG. 2A) andDansyl-F2 (FIG. 2B) for M. tuberculosis. It is to be noted that theordinate extends to 50×10⁸ cfu/ml for F2 (FIG. 2A), whereas the ordinatefor Dansy-F2 extends only to 10×10⁸ cfu/ml (FIG. 2B).

DEFINITIONS

In the context of the present invention and the associated claims, thefollowing terms have the following meanings:

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the term “hydrocarbyl” is a short hand term for anon-aromatic group that includes straight and branched chain aliphaticas well as alicyclic groups or radicals that contain only carbon andhydrogen. Inasmuch as alicyclic groups are cyclic aliphatic groups, suchsubstituents are deemed hereinafter to be subsumed within the aliphaticgroups. Thus, alkyl, alkenyl and alkynyl groups are contemplated,whereas aromatic hydrocarbons such as phenyl and naphthyl groups, whichstrictly speaking are also hydrocarbyl groups, are referred to herein asaryl groups, substituents, moieties or radicals, as discussedhereinafter. An aralkyl substituent group such as benzyl is deemed anaromatic group. A substituent group containing both an aliphatic ringand an aromatic ring portion such as tetralin (tetrahydronaphthalene)that is linked directly through the aliphatic portion to the X group isdeemed a non-aromatic, hydrocarbyl group. On the other hand, a similargroup bonded directly to the X group via the aromatic portion, is deemedto be a substituted aromatic group. Where a specific aliphatichydrocarbyl substituent group is intended, that group is recited; i.e.,C₁-C₄ alkyl, methyl or dodecenyl. Exemplary hydrocarbyl groups contain achain of 1 to about 12 carbon atoms, and preferably 1 to about 8 carbonatoms, and more preferably 1 to 4 carbon atoms.

A particularly preferred hydrocarbyl group is an alkyl group. As aconsequence, a generalized, but more preferred substituent can berecited by replacing the descriptor “hydrocarbyl” with “alkyl” in any ofthe substituent groups enumerated herein.

Examples of alkyl radicals include methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl,octyl, decyl, dodecyl and the like. Cyclic alkyl radicals such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl arealso contemplated, as are their corresponding alkenyl and alkynylradicals. Examples of suitable straight and branched chain alkenylradicals include ethenyl (vinyl), 2-propenyl, 3-propenyl,1,4-pentadienyl, 1,4-butadienyl, 1-butenyl, 2-butenyl, 3-butenyl,decenyl and the like. Examples of straight and branched chain alkynylradicals include ethynyl, 2-propynyl, 3-propynyl, decynyl, 1-butynyl,2-butynyl, 3-butynyl, and the like.

As a skilled worker will understand, a substituent that cannot existsuch as a C₁ alkenyl or alkynyl group is not intended to be encompassedby the word “hydrocarbyl”, although such substituents with two or morecarbon atoms are intended.

Usual chemical suffix nomenclature is followed when using the word“hydrocarbyl” except that the usual practice of removing the terminal“yl” and adding an appropriate suffix is not always followed because ofthe possible similarity of a resulting name to one or more substituents.Thus, a hydrocarbyl ether is referred to as a “hydrocarbyloxy” grouprather than a “hydrocarboxy” group as may possibly be more proper whenfollowing the usual rules of chemical nomenclature.

Illustrative hydrocarbyloxy groups include methoxy, ethoxy, andcyclohexenyloxy groups. On the other hand, a hydrocarbyl groupcontaining a —C(O)— functionality is referred to as a hydrocarboyl(acyl) and that containing a —C(O)O— is a hydrocarboyloxy group inasmuchas there is no ambiguity. Exemplary hydrocarboyl and hydrocarboyloxygroups include acyl and acyloxy groups, respectively, such as acetyl andacetoxy, acryloyl and acryloyloxy.

Amide, ester and thioester links can be formed between an alicyclic,aromatic or heteroaromatic ring containing a C(O) group and a nitrogen,oxygen or sulfur atom, respectively. Similarly, a guanidino linker canbe formed between an alicyclic, aromatic or heteroaromatic ringcontaining a NHC(NH) group and a nitrogen, a urethane, carbonate orthiocarbonate can be formed between an aromatic or heteroaromatic ringcontaining a OC(O) group and a nitrogen, oxygen or sulfur, respectively.A compound containing a urea linker, urethane linker or isothiourealinker [NHC(O)S] can be formed between an alicyclic, aromatic orheteroaromatic ring containing a NHC(O) group and a nitrogen, oxygen orsulfur, respectively.

A “carboxyl” substituent is a —C(O)O H group. A C₁-C₆ hydrocarbylcarboxylate is a C₁-C₆ hydrocarbyl ester of a carboxyl group. Acarboxamide is a —C(O)NR³R⁴ substituent, where the R groups are definedelsewhere. Similarly, a sulfonamide is a —S(O)₂NR³R⁴ substituent, wherethe R groups are defined hereinafter. Illustrative R³ and R⁴ groups thattogether with the depicted nitrogen of a carboxamide form a 5-7-memberedring that optionally contains 1 or 2 additional hetero atoms thatindependently are nitrogen, oxygen or sulfur, include morpholinyl,piperazinyl, oxathiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, pyrazolyl,1,2,4-oxadiazinyl and azepinyl groups.

The term “aryl”, alone or in combination, means a phenyl, naphthyl orother radical as recited hereinafter that optionally carries one or moresubstituents selected from hydrocarbyl, hydrocarbyloxy, halogen,hydroxy, amino, nitro and the like, such as phenyl, p-tolyl,4-methoxyphenyl, 4-(tert-butoxy)phenyl, 4-fluorophenyl, 4-chlorophenyl,4-hydroxyphenyl, and the like. The term “arylhydrocarbyl”, alone or incombination, means a hydrocarbyl radical as defined above in which onehydrogen atom is replaced by an aryl radical as defined above, such asbenzyl, 2-phenylethyl and the like.

The term “arylhydrocarbyloxycarbonyl”, alone or in combination, means aradical of the formula —C(O)—O-arylhydrocarbyl in which the term“arylhydrocarbyl” has the significance given above. An example of anarylhydrocarbyloxycarbonyl radical is benzyloxycarbonyl.

The term “aryloxy” means a radical of the formula aryl-O— in which theterm aryl has the significance given above. The term “aromatic ring” incombinations such as substituted-aromatic ring sulfonamide,substituted-aromatic ring sulfinamide or substituted-aromatic ringsulfenamide means aryl or heteroaryl as defined above.

DETAILED DESCRIPTION OF THE INVENTION

The present invention contemplates a compound, a composition containingan anti-bacterial amount of the compound and an antibacterial method ofusing the compound alone or in conjunction with a synergistic amount ofa) a human cathelicidin antimicrobial peptide LL-37 or b) an antibioticthat targets the cell wall and/or the cell membrane.

Compounds

The present invention contemplates a compound corresponding in structureto structural Formula I or a pharmaceutically acceptable salt of thatcompound

wherein V is O or NR⁹, Y is halogen, OR¹⁰, C₁-C₄ hydrocarbyl or NHR¹⁰, Zis NR²—X—R¹ or CH₂—R⁸, and n is 1-6. In a compound of Formula I, X ishydrido (H), S(O)₂, C(O), C(O)NR⁷, C(NH)NR⁷ or C(O)O, with the provisothat when X is H, R¹ and CH₂—R⁸ are absent. Preferably, X is other thanH, and is S(O)₂ or C(O). R⁹ is hydrido (H) or C₁-C₄ hydrocarbyl; and R¹⁰is hydrido or C₁-C₄ hydrocarbyl. R¹ and R⁸ are the same or different,and each can be an alicyclic, aromatic or heteroaromatic ring systemcontaining one ring or two fused rings each having 5-7 atoms in thering. A heterocyclic ring system can contain one, two, three or fourring atoms that are other than carbon. Such non-carbon ring atoms arenitrogen, sulfur and oxygen [N, S and O].

The ring system can contain up to three substituents (R^(1a), R^(1b),and R^(1c)) other than hydrogen bonded to the ring atoms that themselvescan be the same or different. Each of those three groups, R^(1-c), isseparately selected from the group consisting of C₁-C₆ hydrocarbyl,C₁-C₆ hydrocarbyloxy, C₁-C₆ hydrocarbyloxycarbonyl, trifluoromethyl(—CF₃), trifluoromethoxy (—OCF₃), C₁-C₇ hydrocarboyl (acyl), hydroxy-,halogen, halogen-substituted C₁-C₇ hydrocarboyl, C₁-C₆hydrocarbylsulfonyl, C₁-C₆ hydrocarbyloxysulfonyl, nitro, phenyl,benzyl, cyano, carboxyl, C₁-C₇ hydrocarbyl carboxylate [C(O)O—C₁-C₇hydrocarbyl], carboxamide [C(O)NR³R⁴] or sulfonamide [S(O)₂NR³R⁴]wherein the amido nitrogen in either the carboxamide or sulfonamidegroup has the formula NR³R⁴ in which R³ and R⁴ are the same or differentand are H, C₁-C₄ hydrocarbyl, or R³ and R⁴ together with the depictednitrogen form a 5-7-membered ring that optionally contains 1 or 2additional hetero atoms that independently are nitrogen, oxygen orsulfur, MAr, where M is —CH₂—, —O— or —N═N— and Ar is a single-ringedaryl group, and NR⁵R⁶ wherein R⁵ and R⁶ are the same or different andare H, C₁-C₄ hydrocarbyl, C₁-C₄ acyl, C₁-C₄ hydrocarbylsulfonyl, or R⁵and R⁶ together with the depicted nitrogen form a 5-7-membered ring thatoptionally contains 1 or 2 additional hetero atoms that independentlyare nitrogen, oxygen or sulfur. R² and R⁷ are the same or different andare hydrido (H) or C₁-C₄ hydrocarbyl. In preferred practice, n is 2-4,and more preferably 2.

A particularly preferred compound corresponds in structure to FormulaII, below, or a

pharmaceutically acceptable salt of that compound in which V, X, Y, n,R¹ and R² are as defined above.

In structural Formula II, X is preferably S(O)₂, C(O), C(O)NR⁷, C(NH)NR⁷or C(O)O. V is preferably NH, and Y is preferably halogen, and morepreferably chloro or fluoro. R¹ is an alicyclic, aromatic orheteroaromatic ring system containing one ring or two fused rings eachhaving 5-7 atoms in the ring. A heterocyclic ring system can containone, two, three or four ring atoms that are other than carbon. Suchnon-carbon ring atoms are nitrogen, sulfur and oxygen [N, S and O].

The R¹ ring system can contain up to three substituents (R^(1a), R^(1b),and R^(1c)) other than hydrogen bonded to the ring atoms that themselvescan be the same or different. Each of those three groups, R^(1-c), isseparately selected from the group consisting of C₁-C₆ hydrocarbyl,C₁-C₆ hydrocarbyloxy, C₁-C₆ hydrocarbyloxycarbonyl, trifluoromethyl(—CF₃), trifluoromethoxy (—OCF₃), C₁-C₇ hydrocarboyl (acyl), hydroxy-,halogen, halogen-substituted C₁-C₇ hydrocarboyl, C₁-C₆hydrocarbylsulfonyl, C₁-C₆ hydrocarbyloxysulfonyl, nitro, phenyl,benzyl, cyano, carboxyl, C₁-C₇ hydrocarbyl carboxylate [C(O)O—C₁-C₇hydrocarbyl], carboxamide [C(O)NR³R⁴] or sulfonamide [S(O)₂NR³R⁴]wherein the amido nitrogen in either the carboxamide or sulfonamidegroup has the formula NR³R⁴ in which R³ and R⁴ are the same or differentand are H, C₁-C₄ hydrocarbyl, or R³ and R⁴ together with the depictednitrogen form a 5-7-membered ring that optionally contains 1 or 2additional hetero atoms that independently are nitrogen, oxygen orsulfur, MAr, where M is —CH₂—, —O— or —N═N— and Ar is a single-ringedaryl group, and NR⁵R⁶ wherein R⁵ and R⁶ are the same or different andare H, C₁-C₄ hydrocarbyl, C₁-C₄ acyl, C₁-C₄ hydrocarbylsulfonyl, or R⁵and R⁶ together with the depicted nitrogen form a 5-7-membered ring thatoptionally contains 1 or 2 additional hetero atoms that independentlyare nitrogen, oxygen or sulfur. R² and R⁷ are the same or different andare hydrido (H) or C₁-C₄ hydrocarbyl.

In some preferred embodiments of compounds of one or both of Formulas Iand II, X is S(O)₂, whereas in other preferred embodiments, X is C(O).Thus, in a first such preferred embodiment, a compound is a sulfonamide,whereas in a second preferred embodiment, the compound is a carboxamide.

It is separately preferred that the R¹ (and also R⁸) group be arelatively large group so that R¹ (and R⁸) is preferably a two fusedring system rather than a single ring. Illustrative two fused ringsystems include naphthyl, benzofuranyl, isobenzofuranyl, indoyl,pyrano[3,4-b]pyrrolyl, benzoxazolyl, anthranil, tetralinyl, decalinyl,benzopyryranyl, quinolinyl, isoquinolinyl, cinolinyl, quinazolinyl,pyrido[3,2-b]pyridinyl, purinyl, 1,4,2-benzoxazinyl, thionaphthenyl,isothionaphtheneyl, benzimidazolyl, benzimidazolinyl, benzthiazolyl,benzoxazolyl and the like. It is further preferred that the ring bondeddirectly to X itself be aromatic, whereas the ring to which the aromaticring is fused need not itself also be aromatic, although preferably bothrings are aromatic.

It is additionally preferred that the R¹ group contain at least onesubstituent. One preferred substituent is a NR⁵R⁶ group, where R⁵ and R⁶are both C₁-C₄ hydrocarbyl, such as methyl (C₁) or R⁵ and R⁶ togetherwith the depicted nitrogen form a 5-7-membered ring that contains 1 or 2additional hetero atoms that independently are nitrogen, oxygen orsulfur, such as a N-morpholinyl group.

R² and R⁷ are both preferably hydrido (H).

Illustrative preferred compounds are shown below in Formulas III andIIIB-IIIG.

Methods and Pharmaceutical Compositions

A contemplated compound useful in a method of the invention can beprovided for use by itself, or as a pharmaceutically acceptable salt.Regardless of whether in the form of a salt or not, a contemplatedcomposition is typically dissolved or dispersed in a pharmaceuticallyacceptable diluent or carrier that forms a pharmaceutical compositionand that pharmaceutical composition is administered to the cells inculture or to the cells of a host mammal.

A contemplated pharmaceutical composition contains an amount of acontemplated compound or a pharmaceutically acceptable salt thereofdissolved or dispersed in a physiologically tolerable diluent or carrierthat is effective to provide a bacteriostatic or bactericidal treatment(an antibacterial effective amount) for bacteria. Bacteria contemplatedfor such treatment particularly include Gram positive bacteria such asB. anthracis, B. subtilis, Streptococcus mutans, M. tuberculosis and S.aureus, including the methicillin-susceptible strain, Newman, and theMRSA strain, Sanger 252, and also for Gram negative bacteria such as E.coli or Shigella flexneri having impaired drug efflux systems or whenadministered along with a) the human cathelicidin antimicrobial peptideLL-37 or b) an antibiotic that targets the cell wall and/or the cellmembrane. The bacteria can be treated when present in a cell culture oras an infection in a mammal.

A contemplated pharmaceutical composition can be contacted with(administered or provided to) bacteria or bacterially-infected mammaliancells in vitro as in a cell culture, or in vivo as in an infected,living, host mammal in need.

A contemplated composition is typically administered a plurality oftimes over a period of days to thereby contact the cells to be treated.More usually, a contemplated composition is administered once or twicedaily. It is contemplated that once administration of a contemplatedcompound has begun, the compound will be administered chronically forthe duration of the study being carried out or until the desiredbacterium has been eliminated or its growth slowed or stoppedsufficiently so that a treated mammal's immune system can eliminate theinfection.

A contemplated compound is usually utilized at picomolar to micromolarto millimolar amounts. Thus, an effective amount of a contemplatedcompound present in a contemplated pharmaceutical composition is thatwhich provides a concentration of about 100 picomolar to about 1000micromolar (1 millimolar) to a host animal's blood stream or to an invitro cell medium in practicing a contemplated method of the invention.A more usual amount is about one micromolar to about 500 micromolar. Askilled worker can readily determine an appropriate dosage level of acontemplated compound to inhibit a desired amount of fucosylation and/orsialylation.

A further aspect of the invention contemplates a method of inhibitingthe growth of bacteria, and particularly Gram positive bacteria, thatcomprises the steps of contacting the bacteria with an antibacterialamount of a compound of Formula I or its pharmaceutically acceptablesalt. In one aspect, the bacterium is B. anthracis, whereas in anotheraspect, the bacterium is S. aureus, including the Newman and Sanger 252strains; both being Gram positive bacteria. In further aspects, thebacteria can be M. tuberculosis, B. subtilis or Streptococcus mutans,which are also Gram positive bacteria.

Gram negative mutants whose drug efflux systems have been impaired as bycompounds such as L-phenylalanyl-L-arginyl beta-naphthylamidedihydrochloride (MC-207110), D-Orn-DhPhe-aminoquinoline (MC-02595), and4-aminomethyl-2-pyrrolindinecarboxamide-D-hPhe-6-aminoquinoline(MC-04124), or whose ClpXp protease system is impaired as by a compounddiscussed in inhibition such as E. coli, Shigella flexneri orCaulobacter crescentus in Cheng et al., Protein Sci. 2007 16:1535-1542.

The contacted bacteria can be present in a cell culture or in aninfected mammal. When the latter is the case, the bacteria are contactedby administration of a compound of Formula I to the infected mammal.Particularly when the treated bacteria are present in an infectedmammal, the bacteria are contacted a plurality of times.

In case of any antibacterial method discussed herein, the compound ofFormula I or its pharmaceutically acceptable salt is preferably presentin a pharmaceutical composition as discussed above. A particularlypreferred compound of Formula I such as a compound of Formula III isoften referred to herein as Dansyl-F2 and has the structural formula

Another aspect of the invention contemplates the use of a contemplatedcompound to provide enhanced inhibition of the microbial ClpXP proteaseenzyme, thereby providing enhanced synergistic antimicrobial activitywith cathelicidin antimicrobial peptides and antibiotics that target thecell and/or cell membrane such as penicillin and daptomycin in Grampositive bacteria such as B. anthracis and drug resistant strains of S.aureus. The ClpXP inhibition provided by a contemplated compound istypically enhanced relative to that provided by F2 by a factor of about2 to about 5. Like F2, a contemplated compound can simultaneouslysensitize pathogenic bacteria to host defenses and pharmaceuticalantibiotics.

Thus, use of both 1) a contemplated compound and 2) a) the humancathelicidin antimicrobial peptide LL-37 or b) an antibiotic thattargets the cell wall and/or the cell membrane can kill treated Grampositive bacteria at concentrations of the two active components thatare separately ineffective by themselves to kill the bacteria. A similarsynergistic effect can be obtained with Gram negative bacteria when adrug efflux system-impairing drug such as the before-mentionedL-phenylalanyl-L-arginyl beta-naphthylamide dihydrochloride (MC-207110),D-Orn-D-hPhe-aminoquinoline (MC-02595), and4-aminomethyl-2-pyrrolindinecarboxamide-D-hPhe-6-aminoquinoline(MC-04124). Illustrative cell wall-targeting antibacterials include thebeta-lactam antibacterials like a penicillin (e.g. ampicillin), acephalosporin and a monobactam. Daptomycin is an illustrative cellmembrane targeting antibacterial agent.

In this instance, the concentration of a contemplated compound is aboutone-half or less that of F2 that accomplishes the same result.Illustrative concentrations are about one-half the MIC value determinedfor in vitro cultured bacteria to be killed or whose growth is to beinhibited. Because the two components work together, the amount of eachutilized is referred to a synergistic amount.

Thus, another contemplated method of inhibiting the growth of bacteriathat comprises the steps of contacting the bacteria with a synergisticamount of a compound or its pharmaceutically acceptable salt of FormulaI and a synergistic amount of a) a human cathelicidin antimicrobialpeptide LL-37 or b) an antibiotic that targets the cell wall and/or thecell membrane for Gram positive bacteria, and the same for Gram negativebacteria plus the addition of an effective amount of an effluxsystem-impairing drug, as noted above.

A contemplated pharmaceutical composition can be administered orally(perorally), parenterally, by inhalation spray in a formulationcontaining conventional nontoxic pharmaceutically acceptable carriers,adjuvants, and vehicles as desired. The term parenteral as used hereinincludes subcutaneous injections, intravenous, intramuscular,intrasternal injection, or infusion techniques. Formulation of drugs isdiscussed in, for example, Hoover, John E., Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa.; 1975 and Liberman, H. A. andLachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York,N.Y., 1980.

For injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation can also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that can be employed are water, Ringer's solution,and isotonic sodium chloride solution, phosphate-buffered saline. Liquidpharmaceutical compositions include, for example, solutions suitable forparenteral administration. Sterile water solutions of an activecomponent or sterile solution of the active component in solventscomprising water, ethanol, or propylene glycol are examples of liquidcompositions suitable for parenteral administration.

In addition, sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides.

In addition, fatty acids such as oleic acid find use in the preparationof injectables. Dimethyl acetamide, surfactants including ionic andnon-ionic detergents, polyethylene glycols can be used. Mixtures ofsolvents and wetting agents such as those discussed above are alsouseful.

Sterile solutions can be prepared by dissolving a contemplated compoundin the desired solvent system, and then passing the resulting solutionthrough a membrane filter to sterilize it or, alternatively, bydissolving the sterile compound in a previously sterilized solvent understerile conditions.

Solid dosage forms for oral administration can include capsules,tablets, pills, powders, and granules. In such solid dosage forms, acontemplated compound is ordinarily combined with one or more excipientsappropriate to the indicated route of administration. If administeredper os, the compounds can be admixed with lactose, sucrose, starchpowder, cellulose esters of alkanoic acids, cellulose alkyl esters,talc, stearic acid, magnesium stearate, magnesium oxide, sodium andcalcium salts of phosphoric and sulfuric acids, gelatin, acacia gum,sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, andthen tableted or encapsulated for convenient administration. Suchcapsules or tablets can contain a controlled-release formulation as canbe provided in a dispersion of active compound in hydroxypropylmethylcellulose. In the case of capsules, tablets, and pills, the dosage formscan also comprise buffering agents such as sodium citrate, magnesium orcalcium carbonate or bicarbonate. Tablets, capsules and pills canadditionally be prepared with enteric coatings, and such coatings arepreferred.

A mammal in need of treatment and to which a pharmaceutical compositioncontaining a contemplated compound is administered can be a primate suchas a human, an ape such as a chimpanzee or gorilla, a monkey such as acynomolgus monkey or a macaque, a laboratory animal such as a rat, mouseor rabbit, a companion animal such as a dog, cat, horse, or a foodanimal such as a cow or steer, sheep, lamb, pig, goat, llama or thelike. Where in vitro mammalian cell contact is contemplated, a cultureof cells from an illustrative mammal is often utilized, as isillustrated hereinafter.

Preferably, the pharmaceutical composition is in unit dosage form. Insuch form, the composition is divided into unit doses containingappropriate quantities of the active agent. The unit dosage form can bea packaged preparation, the package containing discrete quantities ofthe preparation, for example, in vials or ampules.

A compound of the invention can be provided for use by itself, or as apharmaceutically acceptable salt. A contemplated compound is an amineand can typically be used in the form of a pharmaceutically acceptableacid addition salt derived from an inorganic or organic acid. Exemplarysalts include but are not limited to the following: acetate, adipate,alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate,butyrate, camphorate, camphorsulfonate, digluconate,cyclopentanepropionate, dodecylsulfate, ethanesulfonate,glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate,fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxy-ethanesulfonate, lactate, maleate, methanesulfonate,nicotinate, 2-naphthalenesulfonate, oxalate, palmoate, pectinate,persulfate, 3-phenylpropionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate, mesylate and undecanoate.

The reader is directed to Berge, J. Pharm. Sci. 68(1):1-19 (1977) forlists of commonly used pharmaceutically acceptable acids and bases thatform pharmaceutically acceptable salts with pharmaceutical compounds.

In some cases, the salts can also be used as an aid in the isolation,purification or resolution of the compounds of this invention. In suchuses, the salt prepared need not be pharmaceutically acceptable.

Compound Synthesis

A contemplated compound is readily prepared using chemistry that shouldbe readily understood by a skilled worker. An illustrative synthesis ofthe intermediate,N-(1-(2-aminoethyl)-1H-tetrazol-5-yl)-3-chlorobenzamide, and thepreferred dansyl derivative are set out hereinafter in Scheme 1.Specific syntheses of several of the compounds prepared and assayedherein are also set out hereinafter along with physical data for the newcompounds. A more general synthesis for additional contemplatedcompounds is illustrated below in Scheme 2.

As will be seen in Scheme 2, sulfonamide linkages are readily formed byreaction of the amine ofN-(1-(2-aminoethyl)-1H-tetrazol-5-yl)-3-chlorobenzamide with a sulfonylhalide such as Compound A in methylene chloride as solvent in thepresence of a base. A carboxamide can similarly be formed by reaction ofan acyl halide such as Compound B with the same starting material. Usingthat same omega-amine starting material and reacting it with anisocyanate-containing compound such as Compound C forms a urea linkage,whereas reaction with a halocarbonate-containing compound such inCompound D forms a urethane linkage.

A general synthetic scheme for ester preparation is shown below asScheme 3, wherein the steps of Scheme 2, above are followed for thelater steps, and in which Y, n, R¹ and R² are as described for FormulaI.

Bactericidal/Bacteristatic Assays

Protocol

Overnight (about 18 hours) cultures of B. anthracis Sterne (pXO1⁺,pXo2⁻), B. subtilis and S. flexneri were grown in plain LB medium. Thefollowing morning cultures were inoculated into a flask containing 25 mlof fresh LB at a starting OD₆₀₀ 0.002. Cultures were then transferredinto pre-designated wells in 96 well plates such that lane A contained95 μl of culture, whereas the rest of the plate contained 50 μl ofculture per well (separate plates were used to analyze the differentstrains).

Five microliters (5 μl) of the highest drug concentrations were added tothe top lane in triplicate from DMSO stocks. The assay plates includeduntreated as well as DMSO controls. Drugs were then serially diluted (2fold dilutions) and the plates were sealed with a thermal seal membraneto prevent evaporation. Plates were incubated at 37° C. for 24 hours.Plates were spun down using a Beckmann S2096 plate rotor on an Allegra®X-22R centrifuge. Growth in plates was then inspected visually and theminimal inhibitory concentration (MIC) determined as the lowestinhibitor concentration that inhibited visible growth. The results ofthis assay are shown below in Table 1.

TABLE 1 MIC (μM) Dansyl- Strain ID F2 F2 CPD D* B. anthracis 31.27.8 >200 B. subtilis 26.1 13.3 >200 S. flexneri 7.8 >200 >200 *See,Compound D, below, after Table 3.

Half Maximal Growth Inhibitory Concentration (IC₅₀) Determination

Protocol

Overnight (about 18 hours) cultures of B. anthracis Sterne (pXO1⁺,pXO2⁻; WT), B. anthracis ΔclpX, B. anthracis ΔclpX+pclpX, WT+pUTE29(empty vector), ΔclpX+pUTE29 and S. flexneri (a Gram negative bacterium)were grown in plain LB medium. The following morning cultures wereinoculated into a flask containing 20 ml of fresh LB at a starting OD₆₀₀0.002. Cultures were then transferred into pre-designated wells in 96well microplates and drugs were titrated at concentrations determinedbased on the MIC with each concentration being analyzed in triplicate toa final volume of 100 μl (separate plates were used to analyze thedifferent strains).

Similarly, the each assay plate included untreated as well as DMSOcontrols. Plates were incubated at 37° C. for 24 hours. Cultures werethen diluted 2-fold with fresh medium and transferred to sterile clearflat bottomed 96 well microplates for OD₆₀₀ readings. OD₆₀₀ was recordedon a SpectraMax® M2 microplate reader (Molecular Devices). Inhibitionwas analyzed as growth in the presence of an inhibitor relative tountreated controls as illustrated in FIG. 1. The results of this assayare shown in Table 2 below.

TABLE 2 IC₅₀ (μM) Strain ID F2 Dansyl-F2 WT B. anthracis 17.6 5.28 ΔclpX27.0 5.93 ΔclpX + pclpX 14.8 6.22 WT + pUTE29 27.5 9.88 ΔclpX + pUTE2922.3 6.28 S. flexneri 5.95 >200

Using similar procedures several further F2 derivative compounds wereassayed for potential bactericidal/bacteriostatic activity against B.anthracis Sterne (pXO1⁺, pXO2⁻) and S. flexneri. The results of thoseassays are shown in Table 3, below. The structures of numberedderivatives are shown hereinafter, with their compound number inparentheses.

TABLE 3 MIC¹ MIC¹ S. flexneri B. anthracis Compound # (μM) (μM) F2 3.1325.0 1 >500 >500 2 250 >500 3 >500 >500 4 >500 >500 5 >500 >5006 >500 >500 7 >500 >500 8 >500 >500 9 >500 >500 10 >500 >500 11 500 >50012 >500 >500 13 >500 >500 ¹MIC values were identical in two separatedeterminations.

Further assays were carried out to determine MIC values using CompoundsF2, Dansyl-F2, A, D and E that are shown in Scheme 1, below, along withCompounds 14, 15, and 16 whose structures are illustrated hereinafter.

These assays were carried out as described for the MIC assays ofTable 1. Mycobacterium smegmatis is useful for the research analysis ofother Mycobacteria species in laboratory studies. M. smegmatis iscommonly used in work on the mycobacterium species due to its being a“fast grower” and non-pathogenic, requiring only a biosafety level 1laboratory. This species shares more than 2000 homologs with M.tuberculosis and shares the same unusual cell wall structure of M.tuberculosis and other mycobacterial species. M. smegmatis is thereforea frequently used model for mycobacterial species. M. smegmatis is aGram positive bacterium. The data from this study are shown below inTable 4.

TABLE 4 MIC (μM) Shigella Bacillus Mycobacterium Compound flexnerianthracis smegmatis F2 3.1 25 25 Dansyl-F2 (E) NA 5.3 NA A NA NA NA D NANA — 14 NA NA — 15 NA NA — 16 NA 400 —

A similar MIC study to those discussed above was carried out with TolCmutants of the Gram negative bacteria E. coli and S. flexneri. TolC isan outer membrane protein that has been implicated in many diversecellular functions, including toxin secretion. [Vakharia et al., JBacteriol 183(23):6908-6916 (2001).] TolC is involved in the secretionof alpha-hemolysin secretion and the TolC mutant studied are defectivein secretion of alpha-hemolysin. The data from that study are shownbelow in Table 5.

TABLE 5 MIC (μM) E. coil S. flexneri Compound TolC TolC F2 12.5 3.12Dansyl-F2 (E) 25 6.25

Inhibition of Spore Germination

Spore germination in B. anthracis, a Gram positive organism, is a keystep to its pathogenesis following infection. Inhibition of thistransformation into vegetative bacteria can be considered as aprophylactic form of treatment that could prevent the onset of thedisease. Consequently, novel inhibitors of spore germination provide aglimmer of hope towards tackling this deadly disease. Such inhibitorscould be used directly to prevent fatality or in combination therapy.

It has been found that F2 and Dansyl-F2 inhibit B. anthracis sporegermination in vitro under physiologic conditions. Thus, cultures ofaliquots from spores that germinated into vegetative cells in theabsence of F2 or dansyl-F2 were treated with 50 μM F2 or 50 μM dansyl-F2and assayed 20 hours after initiation. No vegetative cells were observedin either the F2- or dansyl-F2-treated cultures.

Inhibition of Trans-Translation In Vitro

In vitro trans-translation assays were performed on E. coli extractsusing the PURExpress (New England Biolabs) protein synthesis kit with aDHFR template lacking the stop codon and [³⁵S]-methionine, according tothe manufacturer's instructions, with the addition of 2 μM tmRNA andSmpB. Inhibitor compounds were added to 10 μM final concentration.Tagging efficiency was calculated as the ratio of tagged DHFR to totalDHFR (tagged+untagged) from at least 3 repeats. The % inhibition wascalculated using the control reaction with no inhibitor as 100%. Noinhibition was observed when a DHFR template containing a stop codon wasused, indicating that the inhibitors specifically affecttrans-translation and not translation. The results are shown below inTable 6.

TABLE 6 % inhibition Compound ID in vitro F2 88.0  4 74.9 10 68.1 1157.9 12 44.6 13 71.6 9 (A) 78.9 D 52.3 14 47.8 16 37.0 III (E) 89.1F2 and Dansyl-F2 Inhibit Growth of M. tuberculosis

Auto-luminescent M. tuberculosis (LuxTB) was grown to an OD₆₀₀ of0.0125. The indicated concentrations of F2 and Dansyl-F2 were added tothe mycobacterial cultures and luminescence readings were taken every 24hours using a Tecan Infinite® M200 plate reader. The results are shownin Table 7, below, and indicate an enhanced activity for Dansyl-F2compared to F2 itself.

TABLE 7 IC₅₀ (μM) Day F2 Dansyl-F2 2 292 181 3 226 100 4 115 96.4Bactericidal Activity of F2 and Dansyl-F2 Against M. tuberculosis

Wild-type M. tuberculosis was grown to an OD₆₀₀ of 0.0125 and theindicated concentrations of F2 and Dansyl-F2 were added to themycobacterial cultures. The minimum inhibitory concentration (MIC) of F2and Dansyl-F2 were determined as the minimum concentration of drug thatinhibited visible growth of M. tuberculosis. Colony forming units (CFUs)were determined by plating serial dilutions of cultures onto 7H10 agarplates (Difco) supplemented with 10% OADC (Middlebrook) and 0.5%glycerol. Plates were incubated for 3-4 weeks at 37° C. prior toenumeration of CFUs. This study was carried out by Dr. Paolo Manzanilloand Dr. Jeff Cox at the University of California, San Francisco.

The results of this study are shown in FIG. 2A and FIG. 2B. As will beseen, the results indicate higher activity for Dansyl-F2 than for theparent compound, F2. The MIC for Dansyl-F2 against M. tuberculosis wasfound to be about 50 μM.

Materials

All organic solvents were purchased from VWR International, LLC (Radnor,Pa.) unless otherwise stated. 3-Chlorobenzoic acid,N-(3-dimethyl-aminopropyl)-N′-ethylcarbodiimide (EDC),N-hydroxysuccinimide (NHS), triethylamine (TEA), dichloromethane (DCM),dimethylformamide (DMF), 5-aminotetrazole, biotin, 2-bromo-ethylamine,diisopropylethylamine, Di-tert-butyl dicarbonate, N-hydroxysuccinimideand dimethylsulfoxide-d₆ were purchased from Sigma-Aldrich (St. Louis,Mo.). 1-Propyl-1H-tetrazol-5-amine was purchased from ChemBridgeCorporation (San Diego, Calif.). Silica gel (60 Å, 60-200 μm) waspurchased from VWR International (Bridgeport, N.J.). Thin LayerChromatography (TLC) silica gel (IB-F) plates were purchased from J. T.Baker (Phillipsburg, N.J.). Chloroform-d was purchased from CambridgeIsotope Laboratories (Andover, Mass.).

Abbreviations

DMF—Dimethylformamide; DIPEA—Diisopropyl-ethylamine;EDC—1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide;NHS—N-Hydroxysuccinimide;PyBOP—benzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphate. TFA—Trifluoroacetic acid, DCM—Dichloromethane,

Synthesis of F2

3-Chlorobenzoic acid (250 mg, 1.6 mmol) was added to a bound bottomflask containing a solution of DMF/DCM (1:1) at room temperature. Tothis mixture was added EDC (297 mg, 1.9 mmol) NHS (221 mg, 1.90 mmol).The reaction was stirred for 5 minutes at room temperature to obtain auniform solution. A solution of 1-propyl-1H-tetrazol-5-amine (203 mg,1.6 mmol) in DMF/DCM (1:1) was then added to the flask and the reactioncontinuously stirred at room temperature while monitoring via TLC. After12 hours, the reaction was stopped and evaporated to dryness in vacuo.

Ten ml of aq. NaHCO₃ (0.5 M) was added to the residual material followedby extraction with DOM (3×20 ml). The product was purified via columnchromatography on silica gel and eluted with a CH₂Cl₂/MeOH/TEA(9.8:0.1:0.1) solvent system in 65% yield.

¹H-NMR (400 MHz, CDCl₃): δ0.93 (t, J=7.4, 3H), 1.94 m, 2H), 4.28 (t,J=7.4, 2H), 7.45, (m, 2H), 7.90 (m, 2H), 11.78 (bs, 1H). ¹³1C-NMR (100MHz, CDCl₃): δ 10.6, 21.8, 46.7, 124.8, 127.0, 129.7, 131.1, 134.5,136.6, 161.1, 165.6. MS ESI (+) m/z: calculated for C₁₁H₁₂ClN₅O[M+H]⁺266.1, observed [M+H]⁺266.1.

Synthesis of Dansyl-F2

3-Chloro-N-(1-propyl-1H-tetrazol-5-yl)benzamide (A)

3-Chlorobenzoic acid (1 g, 6.4 mmol) was added to a bound bottom flaskcontaining 30 ml of DMF at room temperature. DIPEA (4.5 ml, 25.6 mmol)and PyBOP (1.1 g, 7.7 mmol) were then added. The resulting mixture wasstirred for an additional 30 minutes at room temperature. A solution of5-aminotetrazole (1.1 g, 7.7 mmol) in DMF was slowly added to the flaskand the reaction stirred continuously for 12 hours. The reaction wasterminated and evaporated to dryness in vacuo.

Ten ml of aq. NaHCO₃ (0.5 M) was added to the dried sample followed bythe addition of 30 ml DCM. The white precipitate that crashed out ofsolution was isolated by vacuum filtration followed by 3 washes with DCMto give the product, a white powder (67% yield).

¹H-NMR (400 MHz, DMSO-d6): δ 6.45 (bs, 1H) 7.60 (t, J=8.0 Hz, 1H), 7.73(d, J=8.0 Hz, 1H), 8.04 (d, J=6.9 Hz, 1H) 8.14 (s, 1H). ¹³C-NMR (100MHz, DMSO-d6): δ 127.1, 128.2, 130.7, 132.7, 133.5, 133.8, 150.3, 164.3.

tert-butyl-2-bromoethylcarbamate (B)

A solution of 2-bromoethylamine hydrobromide (5 g, 24.4 mmol) in 20 mlH₂O was stirred at room temperature in a round bottom flask.Di-tert-butyl dicarbonate (2.8 g, 12.2 mmol) was dissolved in 40 ml DCMand added to the flask over a period of 10 minutes. To the resultingbiphasic mixture was added NaOH (2.0 g, 48.8 mmol) dissolved in 20 mlH₂O. This reaction mixture was stirred vigorously at room temperaturefor 4 hours. The organic layer was then isolated and the aqueous layerextracted once with 20 ml DCM. The combined organic phase was washedonce with H₂O, then with 0.2 M HCl to a pH of about 1 (monitored usingpH paper), and finally with water to a final pH of 6-7 for the aqueouslayer. The resulting organic layer was dried over anhydrous MgSO₄,filtered and evaporated to dryness in vacuo to give colorless oil in 75%yield. [Beylin et al., OPPI Briefs 1987 19:78-80.]

¹H-NMR (400 MHz, CDCl₃): δ 1.40 (s, 9H), 3.44 (m, 4H), 5.05 (bs, 1H).¹³C-NMR (100 MHz, CDCl₃) δ 28.7, 33.0, 41.0, 80.1, 156.0.

3-Chloro-N-(1-(2-(3,3-dimethylbutanamido)ethyl)-1H-tetrazol-5-yl)benzamide(C)

Compound (A) (250 mg; 1.12 mmol), Compound (B) (250 mg; 1.12 mmol) and340 mg (3.36 mmol) of Et₃N were added to a round bottomed flaskcontaining 15 ml of DMF and refluxed overnight (about 18 hours). Thereaction mixture was evaporated to dryness under reduced pressure and 20ml of H₂O added. This aqueous phase was extracted with CH₂Cl₂ (3×30 ml)and the organic layers combined, dried over MgSO₄ and furtherconcentrated. The crude product obtained was purified over silica geland eluted with CHCl₃. To provide a brown oil in 42% yield.

¹H-NMR (400 MHz, CDCl₃): δ 1.43 (s, 1H), 3.68 (t, J=7.6 Hz, 2H), 4.75(t, J=7.6 Hz, 2H), 4.92 (bs, 1H), 7.51 (m, 2H), 7.09 (d, J=5.1 Hz, 1H),7.99 (s, 1H), 9.60 (s, 1H). ¹³C-NMR (100 MHz, CDCl₃) δ 29.1, 3804, 40.6,80.7, 126.7, 127.5, 130.7, 132.1, 132.7, 133.8, 150.3, 157.3, 164.3.

N-(1-(2-aminoethyl)-1H-tetrazol-5-yl)-3-chlorobenzamide (D)

The BOC protected F2-derivative Compound (D) was added to a flaskcontaining a 1:1 solution of CH₂Cl₂/TFA at room temperature. Thissolution was stirred for 5 hours upon which the solvent was evaporatedunder reduced pressure. The residual material was dissolved in CH₂Cl₂and extracted with 0.1 M NaHCO₃, dried over anhydrous MgSO₄ andconcentrated. Flash chromatography was performed using 98:2:0.1%(CH₂Cl₂:MeOH:Et₃N) to yield a brownish oil.

¹H-NMR (400 MHz, CDCl₃): δ 2.92 (bs, 2H), 3.62 (m, 2H), 4.69 (m, 2H),7.48 (m, 2H), 7.11 (d, J=5.1 Hz, 1H), 7.95 (s, 1H), 9.80 (s, 1H).¹³C-NMR (100 MHz, CDCl₃): δ47.3, 51.2, 125.4, 126.9, 129.4, 130.6,132.2, 132.9, 148.7, 159.1

3-chloro-N-(1-(2-(5-(dimethylamino)naphthalene-1-sulfonamido)ethyl)-1H-tetrazol-5-yl)benzamide(E)

To a round bottom flask on ice (0° C.) was added the BOC-deprotectedamine Compound (D) and Et₃N dissolved in CH₂Cl₂. This mixture wasstirred for 5 minutes after which a solution of dansyl chloride inCH₂Cl₂ was added dropwise over a period of 15 minutes.

The resulting reaction mixture was left to warm up to room temperaturegradually and stirred continuously for 12 hours. A saturated solution ofNaHCO₃ was added to the resulting composition that was thereafterextracted with CH₂Cl₂. The combined organic layer was dried overanhydrous MgSO₄ and concentrated in vacuo. Column chromatographyperformed on silica gel and elution with CH₂Cl₂ and a MeOH gradient of1-5% gave a brownish solid in 70% yield.

¹H-NMR (400 MHz, CDCl₃): δ 2.90 (s, 6H), 3.47 (t, J=7.2 Hz, 2H), 4.51(t, J=7.2 Hz, 2H), 6.40 (bs, 1H), 7.54 (m, 4H), 8.07 (m, 4H), 10.6 (bs,1H). ¹³C-NMR (100 MHz, CDCl₃): δ41.6, 45.7, 48.3, 115.6, 119.0, 123.4,123.5, 127.8, 128.8, 129.6, 129.7, 129.8, 130.2, 130.8, 131.2, 131.4,134.2, 140.2, 152.2, 161.3, 171.4. ESI MS (+): m/z [M+H]⁺500.2(observed), calculated for C₂₂H₂₂ClN₇O₃S 499.1.

Representative Synthetic Protocol for Compounds 1-8

N-(1-propyl-1H-tetrazol-5-y1)benzamide (1)

Employing 0.25 g (2.1 mmol) of benzoic acid and 0.26 g (2.1 mmol) of1-propyl-1H-tetrazol-5-amine in the procedure described above forCompound F2 and elution with CH₂Cl₂/MeOH/Et₃N (9.8:0.1:0.1) gave theproduct in 54% yield.

¹H-NMR (400 MHz, CDCl₃): δ 0.98 (t, J=7.4 Hz, 3H), 1.94 (q, J=7.4 Hz,2H), 4.06 (t, J=7.4 Hz, 2H), 7.48 (m, 2H), 7.60 (t, J=6.0 Hz, 1H), 8.10(d, J=7.3 Hz, 2H). ¹³C-NMR (100 MHz, CDCl₃): δ 10.1, 19.5, 43.4, 126.1,128.2 131.1, 134.5, 158.1, 163.8.

3-bromo-N-(1-propyl-1H-tetrazol-5-yl)benzamide (2)

Employing 0.25 g (1.2 mmol) of 3-bromo-benzoic acid and 0.16 g (1.2mmol) of 1-propyl-1H-tetrazol-5-amine in the procedure described aboveand elution with CH₂Cl₂/MeOH/Et₃N (9.8:0.1:0.1) gave the product in 34%yield.

¹H-NMR (400 MHz, CDCl₃): δ 0.99 (t, J=7.5 Hz, 3H), 1.91 (q, J=7.5 Hz,2H), 4.05 (t, J=7.1 Hz, 2H), 7.35 (t, J=7.8 Hz, 1H), 7.73 (d, J=7.9 Hz,1H), 8.02 (d, J=7.8 Hz, 1H).). ¹³C-NMR (100 MHz, CDCl₃): δ 10.3, 23.5,45.1, 121.1, 125.7, 127.7, 130.4, 133.9, 135.6, 159.8, 164.2.

3-fluoro-N-(1-propyl-1H-tetrazol-5-yl)benzamide (3)

Employing 0.25 g (1.8 mmol) of 3-fluoro-benzoic acid and 0.23 g (1.8mmol) of 1-propyl-1H-tetrazol-5-amine in the procedure described aboveand elution with CH₂Cl₂/MeOH/Et₃N (9.8:0.1:0.1) gave the product in 52%yield.

¹H-NMR (400 MHz, CDCl₃): δ 0.89 (t, J=7.2 Hz, 3H), 1.85 (q, J=7.4 Hz,2H), 4.03 (t, J=7.4 Hz, 2H), 7.02 (t, J=6.9 Hz, 1H), 7.26 (m, 1H), 7.58(d, J=7.4 Hz, 1H), 7.69 (s, 1H). ¹³C-NMR (100 MHz, CDCl₃): δ 10.7, 25.1,44.2, 123.5 124.9, 129.07, 131.3, 135.1, 146.2, 161.1, 165.9.

4-(dimethylamino)-N-(1-propyl-1H-tetrazol-5-yl)benzamide (4)

Employing 0.25 g (1.5 mmol) of 4-dimethyl-aminobenzoic acid and 0.19 g(1.5 mmol) of 1-propyl-1H-tetrazol-5-amine in the procedure describedabove and elution with CH₂Cl₂/MeOH/Et₃N (9.6:0.2:0.2) gave the productin 46% yield.

¹H-NMR (400 MHz, CDCl₃): δ1.00 (t, J=7.4 Hz, 3 H), 1.93 (q, J=7.4 Hz,2H), 3.08 (s, 6H), 4.15 (t, J=7.4 Hz, 2H), 6.78 (d, J=8.7 Hz, 2H), 7.99(d, J=8.7 Hz, 2H). ¹³C-NMR (100 MHz, CDCl₃) δ 10.1, 20.7, 31.2, 43.3,114.8, 125.0, 131.1, 157.6, 159.1, 165.6.

4-(diethylamino)-N-(1-propyl-1H-tetrazol-5-yl)-benzamide (5)

Employing 0.30 g (1.6 mmol) of 4-diethyl-aminobenzoic acid and 0.20 mg(1.6 mmol) of 1-propyl-1H-tetrazol-5-amine in the procedure describedabove and elution with CH₂Cl₂/MeOH/Et₃N (9.6:0.2:0.2) gave the productin 56% yield.

¹H-NMR (400 MHz, CDCl₃): δ 1.00 (t, J=7.4 Hz, 3H), 1.21 (t, J=6.9 Hz,3H), 1.90 (q, J=7.4 Hz, 2H), 3.44 (q, J=6.9 Hz, 4H), 4.05 (t, J=7.4 Hz,2H), 6.63 (d, J=9.0 Hz, 2H), 7.94 (d, J=9.0 Hz, 2H). ¹³C-NMR (100 MHz,CDCl₃): δ 10.2, 11.4, 23.6, 39.8, 44.5, 112.6, 121.2, 130.4, 149.7,155.9, 164.8.

3-chloro-N-(1-propyl-1-H-tetrazol-5-yl)benzamide (6)

Employing 0.25 g (1.5 mmol) of 2-amino-3-chlorobenzoic acid and 0.19 g(1.5 mmol) of 1-propyl-1H-tetrazol-5-amine in the procedure describedabove and elution with CH₂Cl₂/MeOH/Et₃N (9.4:0.3:0.3) gave the productin 65% yield.

¹H-NMR (400 MHz, CDCl₃): δ 0.99 (t, J=7.6 Hz, 3 H), 1.87 (q, J=7.6 Hz,2H), 4.03 (t, J=7.4 Hz, 2H), 6.18 (s, 2H), 6.64 (t, J=8 Hz, 1H), 7.48(d, J=8 Hz, 1H), 7.9 (d, J=8 Hz, 1H). ¹³C-NMR (100 MHz, CDCl₃): δ 11.7,22.5, 39.6, 106.3, 116.3, 120.5, 130.0, 135.8, 147.8, 162.6, 169.5.

N-(1-propyl-1H-tetrazol-5-yl)cyclohexane-carboxamide (7)

Employing 0.30 g (2.3 mmol) of cyclohexane carboxylic acid and 0.29 mg(2.3 mmol) of 1-propyl-1H-tetrazol-5-amine in the procedure describedabove and elution with CH₂Cl₂/MeOH/Et₃₁NT (9.3:0.5:0.2) gave the productin 49% yield.

¹H-NMR (400 MHz, CDCl₃): δ 0.97 (t, J=7.3 Hz, 3H), 1.27 (m, 3H), 1.42(m, 2H), 1.65 (m, 3H), 1.92 (m, 3H), 2.30 (m, 1H), 4.14 (t, J=7.1 Hz,2H), 8.82 (bs, 1H). ¹³C-NMR (100 MHz, CDCl₃): δ 11.2, 20.1, 26.3, 30.6,41.2, 45.7, 156.8, 167.2.

N-(1-propyl-1H-tetrazol-5-yl)cyclopentane-carboxamide (8)

Employing 0.30 g (2.6 mmol) of cyclopentane carboxylic acid and 0.33 mg(2.6 mmol) of 1-propyl-1H-tetrazol-5-amine in the procedure describedabove and elution with CH₂Cl₂/MeOH/Et₃N (9.3:0.5:0.2) gave the productin 45% yield.

¹H-NMR (400 MHz, CDCl₃): δ 0.98 (t, 3H), 1.56 (m, 2H), 1.67 (m, 4H),1.77 (m, 2H), 1.88 (m, 4H), 2.60 (m, 1H), 4.10 (t, 2H), 9.42, (bs, 1H).

NMR (100 MHz, CDCl₃): δ 10.6, 21.8, 25.5, 29.6, 43.4, 46.6, 53.8, 172.1,179.0.

Synthesis of 3-chloro-N-(1-propyl-1H-tetrazol-5-yl)benzamide (9)

3-chloro-N-(1-propyl-1H-tetrazol-5-yl)benzamide (9)

See the synthesis of Compound (A), above.

Synthesis of Compounds 10-13

Representative Procedure: 3-chloro-N-phenylbenzamide (10)

To a solution of aniline (178 mg, 1.9 mmol) in 20 mL CH₂Cl₂ at roomtemperature was added NaOH (76.2 mg, 2.9 mmol) in 6 mL water. Thismixture was stirred vigorously for 5 minutes after which 3-chlorobenzoylchloride (500 mg, 2.9 mmol) was added drop-wise while stirring over aperiod of 30 minutes. This reaction mixture was stirred for anadditional 2 hours followed by an acid work-up and extraction withCH₂Cl₂ (3×20 mL). The organic layers were combined, dried over anhydrousMgSO₄. The product was purified via column chromatography on silica geland eluted with a CHCl₃/MeOH/Et₃N (9.8:0.1:0.1) to give white crystalsin 81% yield.

¹H-NMR (400 MHz, CDCl₃): δ 7.18 (m, 1H), 7.38, (m, 3H), 7.51 (d, J=7.6Hz, 1H), 7.63 (dd, J=6.6 Hz, 2H), 7.73 (d, J=7.6 Hz, 1H), 7.85 (bs, 1H),7.93 (m, 1H). ¹³C-NMR (100 MHz, CDCl₃): δ 120.4, 124.9, 125.2, 127.4,129.2, 130.1, 131.9, 135.0, 136.8, 137.6, 164.5.

3-chloro-N-(pyridin-4-yl)benzamide (11)

Employing 0.18 g (1.9 mmol) of 4-amino-pyridine and 0.51 g, (2.9 mmol)of 3-chlorobenzoyl chloride in the procedure described above and elutionwith CHCl₃/MeOH/Et₃N (9.8:0.1:0.1) resulted in white crystals (52%yield).

¹H-NMR (400 MHz, CDCl₃): δ 6.92 (d, J=5.6 Hz, 1H), 7.41, (m, 3H), 7.55(s, 1H), 8.34 (d, J=5.6 Hz, 2H), 8.48 (m, 2H), ¹³C-NMR (100 MHz, CDCl₃):δ 120.5, 125.0, 125.3, 127.5, 129.3, 130.3, 132.0, 161.3.

3-chloro-N-cyclohexylbenzamide (12)

Employing 0.19 g (1.9 mmol) of cyclohexylamine and 0.51 g, (2.9 mmol) of3-chloro-benzoyl chloride in the procedure described above and elutionwith CHCl₃/MeOH/Et₃N (9.8:0.1:0.1) gave white crystals in 70% yield.

¹H-NMR (400 MHz, CDCl₃): δ 1.25 (m, 3H), 1.41 (m, 2H), 1.64 (m, 1H),1.75, (m, 2H), 2.04 (m, 2H), 3.95 (m, 1H), 5.97 (s, 1H), 7.35 (m, 1H),7.44 (d, J=8 Hz, 1H), 7.62 (d, J=8 Hz, 1H), 7.73 (s, 1H). ¹³1C-NMR (100MHz, CDCl₃): δ 24.9, 25.5, 33.2, 48.9, 125.0, 127.2, 129.9, 131.3,134.7, 136.9, 165.3.

3-chloro-N-cyclopentylbenzamide (13)

Employing 0.16 g (1.9 mmol) of cyclopentylamine and 0.51 g, (2.9 mmol)of 3-chloro-benzoyl chloride in the procedure described above andelution with CHCl₃/MeOH/Et₃N (9.8:0.1:0.1) gave white crystals in 92%yield).

¹H-NMR (400 MHz, CDCl₃): δ 1.50 (m, 2H), 1.69 (m, 4H), 2.09 (m, 2H),4.38 (m, 1H), 6.30 (s, 1H), 7.36 (m, 1H), 7.45 (d, J=8 Hz, 1H), 7.62 (d,J=8 Hz, 1H), 7.72 (s, 1H). ¹³C-NMR (100 MHz, CDCl₃): δ 23.6, 33.0, 51.7,124.8, 127.0, 129.7, 131.1, 134.5, 136.6, 165.6.

3-chloro-N-(1-(2-(phenylsulfonamido)ethyl)-1H-tetrazol-5-yl)benzamide(16)

To a round bottomed flask at 0° C. was added Compound 4 (0.059 mmol, 1eq) dissolved in 3 ml DCM and Et₃N (0.179 mmol, 3 eq). Benzenesulfonylchloride (0.079 mmol, 1.2 eq) dissolved in chilled DCM was then addeddropwise over a period of 10 minutes. The reaction was stirred for anadditional 20 minutes at 0° C. before being allowed to gradually warm toroom temperature. The reaction was allowed to proceed for 12 hours atroom temperature.

20 ml of DCM was then added to the crude solution followed by extractionwith 0.1M NaHCO₃. The organic phase was dried over anhydrous Na₂SO₄ andconcentrated in vacuo. The product was purified over silica gel andeluted with CH₂Cl₂:MeOH (1:1) to give a white powder in 70% yield.

¹H-NMR (400 MHz, DMSO-d₆): δ 3.39 (t, J=7.6 Hz, 2H), 4.75 (t, J=7.6 Hz,2H), 7.65 (m, 3H), 7.75 (d, J=7.2 Hz, 1H), 7.83 (d, J=7.9 Hz, 2H), 8.02(m, 2H), 8.49 (s, 1H), 11.63 (s, 1H). ¹³C-NMR (100 MHz, DMSO-d6): 37.4,40.3, 125.6, 127.5, 129.0, 130.2, 131.4, 131.9, 132.2, 134.4, 144.5,156.1, 165.0.

Bacterial Strains and Culture Conditions

B. anthracis Sterne (pXO1+pXO2−) was propagated in Brain-Heart Infusion(BHI) medium (Sigma) and S. aureus strains were propagated in TrypticSoy Broth (Hardy). Genetically modified B. anthracis Sterne strains werepreviously described in McGillivray et al., J Innate Immun 1:494-506(2009). Clinical daptomycin nonsusceptible (DapNS)S. aureus clinicalstrains are described [Jones et al., Antimicrob Agents Chemother52:269-278 (2008)], and DapNS MRSA strain SA32D was derived through invitro passage [Sakoulas et al., Antimicrob Agents Chemother 50:1581-1585(2006)].

Pulse-Chase Assays

The half-life of GFP with the E. coli tmRNA tag (GFP-AA) in vivo isdetermined using pulse chase assays [Keller et al., Science 271:990-993(1996)]. E. coli cultures are grown in M9 minimal medium containing nomethionine or cysteine at 30° C. to OD600=0.6, and GFP-AA expression isinduced by the addition of isopropyl-1-thio-β-D-galactopyranoside (IPTG)to 1 mM. 90 Minutes after induction, cells are labeled with 30 μCi[³⁵S]L-methionine (MP Biomedicals) for 1 minute, and chased with amixture of unlabeled L-methionine and L-cysteine at a finalconcentration of 1.25 mg/ml.

At each time point, 450 μl culture is removed and added to 50 μltrichloroacetic acid, and protein is recovered by centrifugation.Protein pellets are resuspended in 50 μl SDS buffer (10 mM Tris-HCl pH8.0, 1% SDS, 1 mM EDTA), separated by gel electrophoresis on 15% SDSpolyacrylamide gels, and the radiolabeled protein bands are visualizedand quantified using a Typhoon™ imager (GE Healthcare Lifesciences).Half-lives are determined by fitting the plots of band intensity versustime to a single exponential. For cells treated with F2, F2 is added to100 μM final concentration at the time of induction.

Bacterial Survival/Growth Assays

Cultures were grown to early log phase and resuspended to an opticaldensity of 0.4 in PBS. B. anthracis Sterne is diluted 1:10 and S. aureus1:100 in assay specific media to approximately 1×10⁶ cfu/ml or 2×10⁶cfu/ml respectively and are grown for 24 hours at 37° C. under staticconditions. Survival is measured by serial dilution and plating toenumerate surviving cfu/ml. Growth is measured by optical density at awavelength of 600 nM. All studies are done at least 3 times and resultsfrom individual studies are combined and presented as mean+/−SEM.

F2 Resistance

Bacteria are grown overnight (about 18 hours) in the presence of 0 μM F2(DMSO control) or 40 μM F2 in RPMI+5% LB. The next day the overnightcultures are diluted 1:100 (B. anthracis) or 1:1000 (S. aureus) inRPMI+5% LB and the indicated amount of F2 is added. Bacteria are thengrown for an additional 24 hours in static conditions and survivingcfu/ml are enumerated.

Whole Blood Killing Assays

Blood collected from healthy donors (use and procedures approved by theUniversity of California San Diego Human Research Protections Program)is incubated with 105 cfu B. anthracis and 40 μM F2 or vehicle control(DMSO) in a total volume of 500 μL and rotated at 37° C. At 30 minutes,a small aliquot is removed, blood was lysed in water and remainingbacteria are enumerated. Studies are performed using blood from threeindividual donors and results are combined and presented as mean+/−SEM.

Cytotoxicity Assay

HeLa cells are grown in Dulbecco's modified Eagle's medium (Difco)+10%fetal bovine serum (Invitrogen)+1% sodium pyruvate+1%penicillin/streptomycin (Difco). 2×10⁴ Cells per well are plated in 96well plates and grown for 24 hours at 37° C.+5% CO₂. Fresh mediumcontaining the indicated concentration of F2 is added for an additional24 hours. Zero μM F2 contains the equivalent vehicle control (DMSO) atthe highest concentration. Untreated cells are included as a control. 20μl of MTS tetrazolium from the CellTiter™ 96 AQueous One Solution CellProliferation Assay (Promega) is added to each well and incubated for 2hours at 37° C.+5% CO₂ before quantifying absorbance at 490 nm. 100%Viability is set at the absorbance of the untreated cells. Twoindependent studies are combined and results are presented asmean+/−SEM.

Results Inhibition of the ClpXP Protease.

The compound F2 was identified as part of a high-throughput screen forinhibitors of the protein tagging and degradation pathway known astrans-translation in E. coli. F2 inhibited proteolysis of ClpXpsubstrates in vivo using pulse chase assays that monitored the half-lifeof GFP with the E. coli tmRNA tag (GFP-AA). GFP-AA half-life wasincreased from 5.4+/−0.8 minutes to 18.7+/−5.7 minutes in the presenceof F2. In comparison, bacteria lacking clpX had a protein half-life of53.1+/−14 minutes (average half-life values from 3 studies).

ClpX/P are highly conserved among bacterial species [Chandu et al., ResMicrobiol 155:710-719 (2004); Frees et al., Microbiol 63:1285-1295(2007)] and therefore it is likely that a pharmacological inhibitor canfunction in multiple species. Genetic deletion of ClpX increases thesusceptibility of B. anthracis Sterne to LL-37, the human cathelicidinantimicrobial peptide (see [McGillivray et al., J Innate Immun 1:494-506(2009)].

Cathelicidins are critical first line effectors of innate defenseagainst invasive bacterial infection [Nizet et al., Nature 414:454-457(2001)]. It is hypothesized that if F2 could inhibit the ClpXp proteasein B. anthracis Sterne, the parental bacteria would be renderedsusceptible to cathelicidins in a manner similar to the mutant bacteriaharboring a genetic deletion of the clpX gene.

At a dose of 40 μM, F2 alone did not affect B. anthracis Sterne growth.However, in combination with LL-37, growth is inhibited to the samelevel as the ΔclpX mutant.

To examine more precisely the interaction between F2 and LL-37 inkilling B. anthracis Sterne, survival of the bacteria was assessed after24 hour incubation with vehicle control, 40 μM F2 alone, 1.6 μM LL-37alone, or a combination of 40 μM F2+1.6 μM LL-37. Incubation with eithercompound alone had a negligible effect on survival in comparison to thevehicle control, however when the two compounds were combined, recoveryof B. anthracis cfu was below the limit of detection. Similar resultswere seen with murine cathelicidin, CRAMP.

The interaction between F2 and LL-37 was highly significant (two wayANOVA, P<0.0001), indicating a strong synergistic interaction betweenthese two compounds [Slinker, J Mol Cell Cardiol 30:723-731 (1998). F2functions in a dose-dependent manner because increasing amounts of F2led to decreased bacterial survival when the amount of LL-37 is heldconstant.

To determine whether the synergistic effect of F2 and antimicrobialpeptides on the bacteria could potentially be used in therapeutictreatment of infection, the ability of F2 to augment the bactericidalactivity of human whole blood was assayed. Treatment of blood with 40 μMF2 significantly enhanced killing of B. anthracis in whole bloodcompared with the vehicle control. Cytotoxicity in mammalian cells wasnot seen until a dose of 500-1000 μM F2. Because the ClpXp proteaseplays a role in virulence in a number of pathogens, its inhibition couldrepresent a potential therapeutic target for bacterial species besidesB. anthracis.

S. aureus is a major human pathogen and a leading cause of skin, softtissue and bloodstream infections. Of particular concern is the rise ofantibiotic-resistant strains of S. aureus includingmethicillin-resistant S. aureus (MRSA) now epidemic in healthcare andcommunity settings in the U.S. and other developed countries. Loss ofClpXp leads to decreased virulence of S. aureus and results in a numberof shared phenotypes with B. anthracis including loss of hemolytic andproteolytic activity [Frees et al., Mol Microbiol 48:1565-1578 (2003);McGillivray et al., J Innate Immun 1:494-506 (2009)].

It was hypothesized that F2 may function in a similar manner in S.aureus, and F2 activity was assayed against several strains, includingthe methicillin-susceptible strain, Newman, and the MRSA strain, Sanger252. In both cases survival of S. aureus was only slightly reduced whenthe bacteria were incubated for 24 hours with either 10 μM of LL-37 or40 μM of F2 alone. However, combining LL-37 and F2 revealed a strongsynergistic effect. No colonies were recovered for S. aureus Newman andsurvival was reduced by at least 10,000-fold for MRSA Sanger 252. F2exhibits direct antimicrobial activity against B. anthracis Sterne andMRSA Sanger 252. Pre-treatment of bacteria with 40 μM F2 inducesresistance in S. aureus, but not in B. anthracis under the sameconditions.

Loss of ClpX Increases Susceptibility to Conventional Antibiotics.

Deletion of clpX in B. anthracis resulted in increased susceptibility tohost defenses such as antimicrobial peptides and lysozyme that targetcomponents of the cell-envelope including the cell membrane and/or cellwall [McGillivray et al., J Innate Immun 1:494-506 (2009)]. Becausethese bacterial structures are also targets of certain classes ofantibiotics, it was hypothesized that loss of clpX could increasesusceptibility to cell envelope-acting antibiotics.

The parental B. anthracis Sterne, the ΔclpX mutant and the complementedstrain ΔclpX+pclpX were incubated in media with or without 5 μg/ml ofpenicillin, a cell wall-acting antibiotic. It was found that althoughall three strains grew well in media without penicillin, growth of theΔclpX mutant was significantly reduced in the presence of penicillin.Incubation with daptomycin, an antibiotic that disrupts membranepotential and causes cell-wall stress [Muthaiyan et al., AntimicrobAgents Chemother 52:980-990 (2008)], exerted a similar effect.

No viable bacteria were recovered upon incubation of the ΔclpX mutantwith daptomycin, whereas the parental and the complemented strains werenot significantly affected by daptomycin. This effect is not seen withall antibiotics as no differences in growth were seen upon incubationwith either ciprofloxacin, which targets topoisomerase II, orerythromycin, which targets protein synthesis.

Inhibition of ClpXP Increases Antibiotic Sensitivity

Because ClpX was important for resistance to penicillin and daptomycinin B. anthracis Sterne, whether inhibition of the ClpXP protease usingF2 would sensitize B. anthracis Sterne to penicillin or MRSA Sanger 252to daptomycin was next assayed. Bacteria were incubated with vehiclecontrol, F2 alone, antibiotic alone, or a combination of F2+antibiotic.Bacterial survival (cfu/ml) was determined at 24 hours.

In both cases, neither treatment with F2 nor antibiotic alone had asignificant effect on survival. However, a combination of F2 andpenicillin with B. anthracis Sterne or F2 and daptomycin with MRSAsignificantly reduced bacterial survival in a synergistic manner.Although daptomycin is a relatively new antibiotic, resistant strainshave already been reported in clinical practice [Hayden et al., J ClinMicrobiol 43:5285-5287 (2005); Sakoulas et al., J Clin Microbiol46:220-224 (2008)].

Whether F2 could increase daptomycin susceptibility (DapS) indaptomycin-non-susceptible (DapNS)S. aureus strains was queried next.Strains SA0616 (DapS) and SA0701 (DapNS) were isolated from thebloodstream of a patient with daptomycin treatment failure before andafter daptomycin therapy [Sakoulas et al., J Clin Microbiol 46:220-224(2008)]. DapNS MRSA strain SA32D was derived by in vitro passage of DapSMRSA strain SA32 (25). Both SA0701 and SA32D were more resistant todaptomycin killing than their parental strains, SA0616 and SA32respectively. Also, in both cases, treatment of either SA0701 or SA32Dwith F2 decreased their resistance to daptomycin, although in neithercase did it return daptomycin susceptibility to its original levels.

Cellular Localization of Dansyl-F2 in B. anthracis Sterne

An overnight (about 18 hours) culture of B. anthracis Sterne wasinoculated to a starting OD₆₀₀ of 0.02 in fresh LB growth medium. Thisculture was then transferred into plastic tubes each holding 0.5 ml.Dansyl-F2 was added to duplicate tubes to a final concentration of 3 μM.Assay tubes were incubated at 37° C. for 6 hours after which cells werepelleted, washed once with medium without dansyl-F2, and thenresuspended in 100 μl of LB. An aliquot (5 μl) of the thus preparedcomposition was placed on an agar pad for visualization.

Fluorescence images were obtained on a Eclipse 90i microscope (Nikon)using a 60×TIRF N. A. 1.4 oil immersion objective and a Nikon CoolSNAPHQ CCD camera controlled by simple PCi (Compix, Inc.) Those imagesshowed the fluorescent compound within the bacterial cells.

B. anthracis Sterne Spore Preparation

Following the procedures of Alvarez et al., Antimicrob. Agents and Chem.2010 54:5329-5336, an overnight (about 18 hours) B. anthracis Sterneculture was plated on plain LB Agar plates which were then incubated at37° C. for 5 days to obtain bacterial lawns. The resulting lawns werecollected by flooding with sterile ice cold deionized water. Thissuspension was pelleted by centrifugation (4° C.) at 10,000 rpm for 10minutes. Spores were then washed three times with fresh ice colddeionized water, pelleted by centrifugation and resuspended in the samemedium. Spores were incubated at 70° C. for 30 min to kill anyvegetative cells followed by storage at 4° C. in sterile deionizedwater. Spore viability was assessed by plating the heat-treated sporeson plain LB agar plates and monitoring spore germination by microscopy.

Activation of B. anthracis Sterne Spore Germination

B. anthracis Sterne spores were activated and germinated followingliterature procedures. (Alvarez et al., Antimicrob. Agents and Chem.2010 54:5329-5336; and Akochere et al., J. Biol. Chem. 2007282:12112-12118). Thus, prior to starting an assay spore suspensionswere heat activated at 70° C. for 30 minutes. Germination was achievedby resuspension in 50 mM Tris-HC1 [pH 7.5], 10 mM NaCl supplemented withL-alanine (40 μM) and inosine (250 μM). Spore suspensions were analyzedfor auto-germination in the absence of L-alanine and inosine. Novegetative cells were observed under the microscope.

Germination was monitored spectrophotometrically and using microscopy.Spectrophotometrically, loss in light diffraction following addition ofgerminants was indicated by a decrease in optical density at 580 nm.Microscopic analyses were performed on a Nikon Eclipse E600 and wereused to identify the presence of vegetative cells following incubation.Typically, 0.5 ml assay volumes were used with at least duplicateset-ups. Germination was performed in a 37° C. shaker. At a desired timepoint an aliquot (5 ml) was transferred onto an agar pad and visualizedunder the microscope for signs of germination.

Inhibition of B. anthracis Spore Germination

Inhibition of spore germination was assayed using a similar protocol asthat described above. Immediately following addition of the germinants(L-alanine and inosine) into the germination buffer, 500 μl samples weretransferred into tubes containing a pre-calculated concentration of thedesired test compound. Samples were analyzed at a desired time point0-48 hours by microscopy.

Each of the patents and articles cited herein is incorporated byreference.

The foregoing description and the examples are intended as illustrativeand are not to be taken as limiting. Still other variations within thespirit and scope of this invention are possible and will readily presentthemselves to those skilled in the art.

1. A compound corresponding in structure to structural Formula I or apharmaceutically acceptable salt of that compound

wherein n is 1-6; V is O or NR⁹; R⁹ is hydrido (H) or C₁-C₄ hydrocarbyl;Z is NR²—X—R¹ or CH₂—R⁸; X is hydrido (H), S(O)₂, C(O), C(O)NR⁷,C(NH)NR⁷ or C(O)O, with the proviso that when X is H, R¹ and CH₂—R⁸ areabsent; Y is halogen, OR¹⁰, C₁-C₄ hydrocarbyl or NHR¹⁰; R¹⁰ is hydridoor C₁-C₄ hydrocarbyl; R¹ and R⁸ are the same or different and are analiphatic, aromatic or heteroaromatic ring system containing one ring ortwo fused rings each having 5-7 atoms in the ring, said ring systemcontaining up to three substituents other than hydrogen that themselvescan be the same or different (R^(1a), R^(1b), and R^(1c)), and whereineach of those three groups, R^(1a-c), is separately selected from thegroup consisting of C₁-C₆ hydrocarbyl, C₁-C₆ hydrocarbyloxy, C₁-C₆hydrocarbyloxycarbonyl, trifluoromethyl, trifluoromethoxy, C₁-C₇hydrocarboyl, hydroxy-, halogen, halogen-substituted C₁-C₇ hydrocarboyl,C₁-C₇ hydrocarbylsulfonyl, C₁-C₆ hydrocarbyloxysulfonyl, nitro, phenyl,benzyl, cyano, carboxyl, C₁-C₇ hydrocarbyl carboxylate, carboxamide orsulfonamide wherein the amido nitrogen in either group has the formulaNR³R⁴ in which R³ and R⁴ are the same or different and are H, C₁-C₄hydrocarbyl, or R³ and R⁴ together with the depicted nitrogen form a5-7-membered ring that optionally contains 1 or 2 additional heteroatoms that independently are nitrogen, oxygen or sulfur, MAr, where M is—CH₂—, —O— or —N═N— and Ar is a single-ringed aryl group, and NR⁵R⁶wherein R⁵ and R⁶ are the same or different and are H, C₁-C₄hydrocarbyl, C₁-C₄ acyl, C₁-C₄ hydrocarbylsulfonyl, or R⁵ and R⁶together with the depicted nitrogen form a 5-7-membered ring thatoptionally contains 1 or 2 additional hetero atoms that independentlyare nitrogen, oxygen or sulfur; and R² and R⁷ are the same or differentand are hydrido or C₁-C₄ hydrocarbyl.
 2. The compound orpharmaceutically acceptable salt according to claim 1, wherein n is 2-4.3. The compound or pharmaceutically acceptable salt according to claim1, wherein Z is NR²—X—R¹.
 4. The compound or pharmaceutically acceptablesalt according to claim 3, wherein said compound corresponds instructure to Formula II

in which V, X, Y, n, R¹ and R² are as defined above.
 5. A compoundcorresponding in structure to structural Formula IIA or apharmaceutically acceptable salt of that compound

wherein X is S(O)₂, C(O), C(O)NR⁷, C(NH)NR⁷ or C(O)O; R¹ is analiphatic, aromatic or heteroaromatic ring system containing one ring ortwo fused rings each having 5-7 atoms in the ring, said ring systemcontaining up to three substituents other than hydrogen that themselvescan be the same or different (R^(1a), R^(1b), and R^(1c)), and whereineach of those three groups, R^(1a-c), is separately selected from thegroup consisting of C₁-C₆ hydrocarbyl, C₁-C₆ hydrocarbyloxy, C₁-C₆hydrocarbyloxycarbonyl, trifluoromethyl, trifluoromethoxy, C₁-C₇hydrocarboyl, hydroxy-, halogen, halogen-substituted C₁-C₇ hydrocarboyl,C₁-C₆ hydrocarbylsulfonyl, C₁-C₆ hydrocarbyloxysulfonyl, nitro, phenyl,benzyl, cyano, carboxyl, C₁-C₇ hydrocarbyl carboxylate, carboxamide orsulfonamide wherein the amido nitrogen in either group has the formulaNR³R⁴ in which R³ and R⁴ are the same or different and are H, C₁-C₄hydrocarbyl, or R³ and R⁴ together with the depicted nitrogen form a5-7-membered ring that optionally contains 1 or 2 additional heteroatoms that independently are nitrogen, oxygen or sulfur, MAr, where M is—CH₂—, —O— or —N═N— and Ar is a single-ringed aryl group, and NR⁵R⁶wherein R⁵ and R⁶ are the same or different and are H, C₁-C₄hydrocarbyl, C₁-C₄ acyl, C₁-C₄ hydrocarbylsulfonyl, or R⁵ and R⁶together with the depicted nitrogen form a 5-7-membered ring thatoptionally contains 1 or 2 additional hetero atoms that independentlyare nitrogen, oxygen or sulfur; and R² and R⁷ are the same or differentand are hydrido or C₁-C₄ hydrocarbyl.
 6. The compound orpharmaceutically acceptable salt according to claim 5, wherein X is C(O)or S(O)₂.
 7. The compound or pharmaceutically acceptable salt accordingto claim 6, wherein R¹ is an aromatic or heteroaromatic ring system. 8.The compound or pharmaceutically acceptable salt according to claim 7,wherein said aromatic or heteroaromatic ring system contains one ring ortwo fused rings that contain up to three substituents other thanhydrogen, which substituents can themselves be the same or different. 9.The compound or pharmaceutically acceptable salt according to claim 6,wherein R² is hydrido.
 10. The compound or pharmaceutically acceptablesalt according to claim 6, wherein said compound of Formula IIA has thestructural formula


11. A pharmaceutical composition that comprises a bactericidal orbacteriostatic amount of a compound or its pharmaceutically acceptablesalt of claim 1 dissolved or dispersed in a pharmaceutically acceptablediluent.
 12. A method of inhibiting the growth of bacteria thatcomprises the steps of contacting said bacteria with an antibacterialamount of a compound or its pharmaceutically acceptable salt of claim 1.13. The method according to claim 12, wherein said bacteria arecontacted a plurality of times.
 14. The method according to claim 12,wherein said bacteria are Gram positive.
 15. The method according toclaim 14, wherein said Gram positive bacteria are B. anthracis.
 16. Themethod according to claim 14, wherein said Gram positive bacteria are S.aureus.
 17. The method according to claim 17, wherein said Gram positivebacteria are M. tuberculosis.
 18. The method according to claim 12,wherein said bacteria are Gram negative.
 19. The method according toclaim 12 wherein said bacteria are present in a cell culture.
 20. Themethod according to claim 12 wherein said bacteria are present in aninfected mammal and said bacteria are contacted by administration ofsaid compound to said infected mammal.
 21. The method according to claim12, wherein said compound has the structural formula


22. A method of inhibiting the growth of bacteria that comprises thesteps of contacting said bacteria with a synergistic amount of acompound or its pharmaceutically acceptable salt of claim 1 and asynergistic amount of a) a human cathelicidin antimicrobial peptideLL-37 or b) an antibiotic that targets the cell wall and/or the cellmembrane.
 23. The method according to claim 22, wherein said bacteriaare Gram positive.
 24. The method according to claim 22, wherein saidbacteria are Gram negative and an effective amount of an effluxsystem-impairing drug.
 25. The method according to claim 22, whereinsaid compound has the structural formula